METHOD FOR DETECTING CANCER USING 5-HYDROXYMETHYLCYTOSINE (5-hmC)

ABSTRACT

Disclosed herein is a method which includes extracting genomic deoxyribonucleic acid (DNA) at locations at or near cancer hotspots from a subject, modifying Tier-1 5hmC on the DNA to a modified 5hmC, detecting and identifying the presence or absence of the modified 5hmC, quantifying the detected and identified modified 5hmC; and providing a report comprising a score, wherein the score is indicative of the presence of cancer.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of non-provisional patent application titled “Method For Detecting Cancer Using 5-Hydroxymethylcytosine (5-hmC)”, application Ser. No. 17/577,033, filed in the United States Patent and Trademark Office on Jan. 17, 2022. The specification of the above referenced patent application is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present disclosure relates to a method for detecting cancer. More particularly, it relates to a method for detecting, screening or predicting a likelihood of cancer using specific genomic 5-hydroxymethylcytosine (5hmC) sites at or near cancer mutation hot spots.

BACKGROUND

Cancer is a major disease worldwide. Each year, tens of millions of people are diagnosed with cancer around the world, and more than half of the patients eventually die from it. In many countries, cancer ranks the second most common cause of death following cardiovascular diseases. Early detection of cancer in a person improves the cure and outcomes for many types of cancers.

Efforts in using mutation hotspots as cancer biomarkers have not been fully successful due to the fact that cancer is usually associated with many mutations. These hotspots often do not show up in the majority of cancer cases. No single hotspot is prevalent enough to be used as a universal sensitive cancer marker. Universal markers like methylated cytosine (5-methylcytosine or 5mC) and Tumor Mutation Burden (TMB) have been widely explored as simple markers. However, both markers still lack large-scale validation, precluding implementation in clinical practice.

Mammalian deoxyribonucleic acid (DNA) contains oxidized forms of 5-methylcytosine (5mC). The base 5-hydroxymethylcytosine (5hmC) is the most commonly occurring oxidation product. In one well known mechanism, 5hmC is produced from 5mC in an enzymatic pathway involving three 5mC oxidases, Ten-eleven translocation (TET)1, TET2, and TET3. Formation of 5hmC from 5mC lowers the levels of 5mC genome. The conversion of 5mC to 5hmC may be the first step in a pathway leading towards DNA demethylation. However, the biological role of 5hmC is still unclear, and there may be conflicting results on inhibition of TET and suppressed hydroxymethylation (5hmC), such as promoting somatic cell reprogramming, increased gene expression of tumor suppression, and reduced cholangiocarcinoma progression.

Studies on the functional role of 5hmC have been heavily focused on change in chromosome-wide global 5hmC density or concentration, or regulation of transcription in the promoter region, or loss of 5hmc across many types of cancer. Unlike the uniform distribution of 5mC outside of the promoter regions, satellites, and repeat DNA sequences, 5hmC has distinct distributions across different functional regions, and its abundance varies across different tissues and cell types. Tissue type plays a dominant role in determining the distribution patterns of 5hmC. 5hmC is enriched primarily in the distal regulatory regions, gene bodies of actively expressing genes and promoters, indicating its connection with active transcription. Genome-wide analysis of 5mC has indicated the global hypo-methylation pattern in tumor tissues, whereas depletion of 5hmC has also been associated with the hyper-methylation of gene bodies in various cancers. Significant enrichment of 5hmC is observed in both tissue-specific and cancer-specific differentially methylated regions as compared with that of 5mC.

Using massive parallel sequencing technique, thousands of genes from pancreatic cancer patients were simultaneously studied in which 5hmC is differentially expressed. Hundreds of genes related to pancreatic development or cancer were found to carry many 5hmC sites. By measuring signal (“peaks’) from thousands of 5hmC all together, “global” 5hmC profiles or patterns in either increase or decrease were observed at chromosomal or at clusters of gene sequence level. For example, the size of the group was described as “log [counts per million (base pair)] on 320 genes, a subset of the 13,180 genes that exhibited a statistically significant (FDR=0.05) increase or decrease in 5hmC”. Even though sample genes and their genomic locations are listed based on filtering criteria, each gene was covered by a few thousand base pair sequence, without pointing out which specific, individual 5hmC sites. However, there is no identification of specific individual 5hmC sites linked to cancer or hotspot mutations linked to cancer. But rather it was assumed the individual hydroxymethylation biomarkers may not have significant individual significance in the evaluation of a pancreatic lesion.

In our study, we demonstrated that, after chemical treatment to convert it to uracil (read as Thymine in NGS sequencing), 5hmCs are detected within CpG islands located either at or near a cancer mutation hotspot (within an 80 bp flanking region). 5hmC detected on these discrete CpG sites showed a significantly greater proportion of cancer versus normal cells. The results showed that the 5hmCs detected at or near caner mutation hotspots consist near entirely by two characteristically distinct 5hmC groups: Tier 1 Group: the cytosine (C) residues that exhibit 3 to 8-fold more likelihood of 5hmCs detected in gDNAs from tumor-cells than from normal-cells; Tier 2 group: equal allele frequency (AF) of 5hmc detected in both normal and tumor-cells. It was hypothesized that, the Tier 1 group of 5hmC is associated with cancer cells and cancer hotspot formation. The 5hmC is an intermediate or precursor before the eventual C to T or G to A mutation. Unlike previous studies looking at the “global” 5hmC signals or patterns of 5hmC (as a group) across large chromosomal region, this study is based on identified specific, individual 5hmC sites at or near known cancer hotspots that display higher 5hmC occurrence in cancer cells. Tier 1 sites individually or combinedly detected can serve as specific marker for cancer. In Tier 2 5hmC sites, both cancer and normal cells have similar level of 5hmC. Tier 2 sites are not good as marker to distinguish between cancer and normal cells.

The detection of these specifically selected, individual Tier-1 5hmC sites at or near hotspot CpG sites in cancer cell can be a more convenient, more direct, and more sensitive cancer detection method than analysing the methylation profile at chromosomal level or from hundreds of sequences of entire genes.

Thus, there is a need for methods for detecting cancer using these specifically located 5hmCs directly at specific base (C or G) resolution.

SUMMARY OF THE INVENTION

A method is disclosed to detect risk of cancer. The method includes extracting genomic deoxyribonucleic acid (DNA) from locations at or near cancer hotspots from a subject, modifying the specific Tier-1 5-hydroxymethylcytosine (5hmC) on the DNA to a modified specific Tier-1 5hmC, detecting and identifying presence or absence of modified Tier-1 5hmC, quantifying the detected and identified modified specific Tier-1 5hmC, and providing a report comprising a score, wherein the score is indicative of the likelihood of a status, a degree, or a severity of the risk of cancer, wherein the specific Tier-1 exist in cancer cell lines, in transformed and immortalized cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C and 2A-2D illustrate examples of individual 5hmC sites as specific cancer marker (Tier 1) or not as marker (Tier 2).

FIG. 3 illustrates average AF % of detected C>T (G>A) at hotspots before and after DNA treatment.

FIG. 4 illustrates 5hmC sites in tumor as percentage of 5hmC in normal at increasing AF cut-off.

FIG. 5 illustrates an example amplification plot from qPCR.

DETAILED DESCRIPTION OF THE INVENTION

Before explaining at least one embodiment of the disclosure in detail, it is to be understood that the disclosure is not necessarily limited in its application to the details set forth in the following description. The disclosure is capable of other embodiments, and of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

As used herein, a cancer mutation hot spot is any single nucleotide having C-to-T or G-to-A substitution mutations reported in the literature that is associated with any cancer. A C>T or G>A change at hotspot resulted in an amino acid change, such as ATM p.R337C, SMARCA4 p.T790M, IDH1 p.R137H, KRAS p.G12C, etc. By way of example, hotspots comprise the following (Table 1):

TABLE 1 Number of Chromo- Position Position affected Gene HGVS.p* some (start) (end) Substitutions^(†) samples AACS p.P495H chr12 125,129,395 125,129,395 C > A 1 AADAT p.L84F chr4 170,087,233 170,087,233 C > A 1 AAK1 p.E341D chr2 69,525,065 69,525,065 C > A 1 AAMP p.E57D chr2 218,269,485 218,269,485 C > A 1 ABCA10 p.E1521D chr17 69,148,896 69,148,896 C > A 1 ABCA2 p.K2310N chr9 137,008,951 137,008,951 C > A 1 ABCA2 p.W750C chr9 137,017,654 137,017,654 C > A 1 ABCB1 p.E686D chr7 87,544,829 87,544,829 C > A 1 ABCB6 p.R177M chr2 219,218,144 219,218,144 C > A 1 ABCB8 p.P68H chr7 151,033,712 151,033,712 C > A 1 ABCC10 p.L657I chr6 43,438,721 43,438,721 C > A 1 ABCC10 p.P724H chr6 43,442,998 43,442,998 C > A 1 ABCC3 p.H93N chr17 50,656,756 50,656,756 C > A 1 ABCC3 p.P1109Q chr17 50,676,536 50,676,536 C > A 1 ABCD4 p.S307I chr14 74,292,764 74,292,764 C > A 1 ABHD13 p.P32H chr13 108,229,313 108,229,313 C > A 1 ABHD15 p.G69V chr17 29,566,761 29,566,761 C > A 1 ABU p.R490L chr10 26,748,628 26,748,628 C > A 1 ABU p.E424D chr10 26,751,677 26,751,677 C > A 1 AASDH p.L440fs chr4 56,354,102 56,354,102 C > CA 5 ABLIM1 p.C106fs chr10 114,601,889 114,601,889 C > CA 1 ACO2 p.I742fs chr22 41,528,493 41,528,493 C > CA 1 ADD3 p.W364fs chr10 110,122,233 110,122,233 C > CA 1 ADD3 p.E670fs chr10 110,133,591 110,133,591 C > CA 1 AGGF1 p.N681fs chr5 77,063,141 77,063,141 C > CA 2 AGL p.N1304fs chr1 99,912,471 99,912,471 C > CA 2 AGTPBP1 p.L1104fs chr9 85,575,386 85,575,386 C > CA 1 ANKRD12 p.N1526fs chr18 9,257,837 9,257,837 C > CA 1 ANO10 p.G229fs chr3 43,577,169 43,577,169 C > CA 1 AP4B1 p.C320fs chr1 113,900,059 113,900,059 C > CA 1 APAF1 p.N591fs chr12 98,677,428 98,677,428 C > CA 4 APC p.K2051fs chr5 112,841,737 112,841,737 C > CA 1 ARCN1 p.A180fs chr11 118,583,892 118,583,892 C > CA 1 ARF1 p.E17fs chr1 228,097,156 228,097,156 C > CA 1 ARFGAP3 p.A153fs chr22 42,834,262 42,834,262 C > CA 1 ARHGAP18 p.L626fs chr6 129,580,092 129,580,092 C > CA 1 ARHGAP19 p.V46fs chr10 97,266,046 97,266,046 C > CA 1 ARHGAP5 p.P193fs chr14 32,091,238 32,091,238 C > CA 1 ARID1B p.N1782fs chr6 157,206,517 157,206,517 C > CA 1 AR1H2 p.H129fs chr3 48,964,975 48,964,975 C > CA 1 CEP162 p.D407fs chr6 84,186,514 84,186,514 C > CAA 1 UBA52 p.K114dup chr19 18,575,096 18,575,096 C > CAAG 1 ATP6V1C2 p.S316fs chr2 10,777,692 10,777,692 C > CAG 3 DSP p.E1778fs chr6 7,581,510 7,581,510 C > CAG 1 FIP1L1 p.E488fs chr4 53,453,080 53,453,080 C > CAG 4 LMO7 p.N937fs chr13 75,841,170 75,841,170 C > CAG 1 RB1 p.A74fs chr13 48,307,352 48,307,352 C > CAG 2 APOBR p.E183dup chr16 28,495,573 28,495,573 C > CAGG 1 CCDC97 p.E245dup chr19 41,319,792 41,319,792 C > CAGG 1 NHSL1 p.P938dup chr6 138,431,542 138,431,542 C > CAGG 1 SERAC1 p.V77fs chr6 158,150,489 158,150,489 C > CAT 2 H1FX p.M1fs chr3 129,315,900 129,315,900 C > CATGGT 1 FGFRE1 p.S486fs chr4 1,025,266 1,025,266 C > CCACA 2 EDC4 p.S617dup chr16 67,879,863 67,879,863 C > CCAG 2 MED15 p.Q218dup chr22 20,564,628 20,564,628 C > CCAG 3 RAI1 p.Q291dup chr17 17,793,779 17,793,779 C > CCAG 1 ATN1 p.Q488_ chr12 6,936,716 6,936,716 C > CCAGCAA 1 Q489dup ATR p.Y239_ chr3 142,562,683 142,562,683 C > CCATACTCTA 1 G240insVEY DCTN1 p.E218dup chr2 74,371,166 74,371,166 C > CCCT 1 BRD7 p.D278fs chr16 50,334,766 50,334,766 C > CCT 4 KIAA0907 p.D517fs chr1 155,916,630 155,916,630 C > CCT 1 RNF43 p.E79dup chr17 58,415,339 58,415,339 C > CCTT 1 ASUN p.R573Q chr12 26,913,544 26,913,544 C > T 1 ASXL1 p.A221V chr20 32,429,997 32,429,997 C > T 1 ASXL1 p.R417X chr20 32,433,447 32,433,447 C > T 1 ASXL1 p.R1415X chr20 32,436,955 32,436,955 C > T 1 ASXL2 p.G79R chr2 25,806,246 25,806,246 C > T 1 ATAD1 p.R201H chr10 87,776,409 87,776,409 C > T 1 ATAD2 p.C387Y chr8 123,359,683 123,359,683 C > T 1 ATAD2 p.R313H chr8 123,369,169 123,369,169 C > T 1 ATAD2B p.R514H chr2 23,857,442 23,857,442 C > T 1 ATAD5 p.S634L chr17 30,835,982 30,835,982 C > T 1 ATAD5 p.P1630L chr17 30,893,742 30,893,742 C > T 1 ATE1 p.A502T chr10 121,743,733 121,743,733 C > T 1 ATE1 p.G236S chr10 121,902,498 121,902,498 C > T 1 ATF2 p.R342H chr2 175,093,221 175,093,221 C > T 1 ATF4 p.A98V chr22 39,521,839 39,521,839 C > T 1 ATF5 p.R226C chr19 49,932,919 49,932,919 C > T 1 ATF6 p.R376X chr1 161,821,100 161,821,100 C > T 2 ATF6B p.V168I chr6 32,121,325 32,121,325 C > T 1 ATF7 p.R152H chr12 53,534,607 53,534,607 C > T 1 ATF7IP p.P209L chr12 14,424,541 14,424,541 C > T 1 ATF7IP p.A347V chr12 14,424,955 14,424,955 C > T 1 A1CF p.R125C chr10 50,836,305 50,836,305 G > A 1 A1CF p.R21C chr10 50,859,880 50,859,880 G > A 1 A2ME1 p.A759T chr12 8,851,824 8,851,824 G > A 1 A4GALT p.R213C chr22 42,693,315 42,693,315 G > A 1 AACS p.R107Q chr12 125,076,573 125,076,573 G > A 1 AACS p.G542D chr12 125,134,799 125,134,799 G > A 1 AACS p.E580K chr12 125,136,721 125,136,721 G > A 1 AAK1 p.T241M chr2 69,530,641 69,530,641 G > A 1 AAMDC p.R69Q chr11 77,869,795 77,869,795 G > A 1 AAR2 p.R207H chr20 36,240,488 36,240,488 G > A 1 AARS2 p.A933V chr6 44,300,707 44,300,707 G > A 1 AARS2 p.R521X chr6 44,305,072 44,305,072 G > A 2 AARS2 p.A15V chr6 44,313,280 44,313,280 G > A 1 AARSD1 p.R171X chr17 42,956,439 42,956,439 G > A 1 AASS p.R910X chr7 122,076,542 122,076,542 G > A 1 AATF p.R474Q chr17 37,019,027 37,019,027 G > A 1 AATK p.R1124C chr17 81,120,257 81,120,257 G > A 1 AATK p.A1101V chr17 81,120,325 81,120,325 G > A 2 AATK p.A408V chr17 81,122,404 81,122,404 G > A 1 ABAT p.A246T chr16 8,768,893 8,768,893 G > A 1 ABCAI p.R1839C chr9 104,793,292 104,793,292 G > A 1 CCDC175 p.N632fs chr14 59,525,381 59,525,381 G > GTT 1 AAAS p.L128M chr12 53,315,352 53,315,352 G > T 1 AAGAB p.H204N chr15 67,209,470 67,209,470 G > T 1 AAKI p.P358H chr2 69,520,971 69,520,971 G > T 1 AASDHPPT p.E299X chr11 106,096,872 106,096,872 G > T 1 ABCA2 p.P1893Q chr9 137,011,528 137,011,528 G > T 1 ABCA5 p.S1190Y chr17 69,260,408 69,260,408 G > T 1 ABCA7 p.R1128M chr19 1,053,491 1,053,491 G > T 1 ABCA7 p.G1374C chr19 1,055,266 1,055,266 G > T 1 ABCB10 p.A647D chr1 229,521,602 229,521,602 G > T 1 ABCF3 p.R546L chr3 184,192,668 184,192,668 G > T 1 ABHD14B p.L69M chr3 51,971,466 51,971,466 G > T 1 ABI2 p.K428N chr2 203,427,208 203,427,208 G > T 1 ABU p.R1130M chr9 130,885,679 130,885,679 G > T 1 ABL1M3 p.E247D chr5 149,217,030 149,217,030 G > T 1 ACAD8 p.K345N chr11 134,261,833 134,261,833 G > T 1 ACAP3 p.A565D chr1 1,295,747 1,295,747 G > T 1 ACAT1 p.R21M chr11 108,121,668 108,121,668 G > T 1 ACBD4 p.G106C chr17 45,137,040 45,137,040 G > T 1 ACIN1 p.L141M chr14 23,090,591 23,090,591 G > T 1 ACOT1 p.K349N chr14 73,543,436 73,543,436 G > T 1 SNCAIP p.E384fs chr5 122,425,498 122,425,500 TGA > T 1 TRIP11 p.S1662fs chr14 91,995,420 91,995,422 TGA > T 1 UBAP2 p.S699fs chr9 33,933,499 33,933,501 TGA > T 1 ZNF263 p.R544fs chr16 3,290,126 3,290,128 TGA > T 1 AFAP1Ll p.K694del chr5 149,332,797 149,332,800 TGAA > T 1 AKAP8 p.F473del chr19 15,360,955 15,360,958 TGAA > T 1 BCR p.K817del chr22 23,287,193 23,287,196 TGAA > T 1 CEP250 p.K1467del chr20 35,502,763 35,502,766 TGAA > T 1 CIC p.K1340del chr19 42,293,809 42,293,812 TGAA > T 1 FAM102B p.K6del chr1 108,560,433 108,560,436 TGAA > T 4 GPATCH11 p.E222del chr2 37,094,135 37,094,138 TGAA > T 1 GSPT2 p.E502del chrX 51,745,120 51,745,123 TGAA > T 1 KAT6B p.E1073del chr10 75,022,066 75,022,069 TGAA > T 2 NAPA p.F207del chr19 47,492,059 47,492,062 TGAA > T 1 PAF1 p.F313del chr19 39,388,385 39,388,388 TGAA > T 1 PAIP2 p.E42del chr5 139,363,901 139,363,904 TGAA > T 1 PHB2 p.F52del chr12 6,970,251 6,970,254 TGAA > T 1 PLK1 p.S471del chr16 23,689,385 23,689,388 TGAA > T 1 POLR3GL p.E187del chr1 145,978,078 145,978,081 TGAA > T 1 RANBP2 p.E2903del chr2 108,781,372 108,781,375 TGAA > T 1 RBM33 p.E210del chr7 155,700,823 155,700,826 TGAA > T 2 SYBU p.S174del chr8 109,586,065 109,586,068 TGAA > T 3 USP37 p.S904del chr2 218,457,093 218,457,096 TGAA > T 1 SH3GLB2 p.V257del chr9 129,009,838 129,009,841 TGAC > T 1 UBE3C p.D1062del chr7 157,267,686 157,267,689 TGAC > T 1 URU p.D311del chr19 30,009,236 30,009,239 TGAC > T 5 SNRNP70 p.D375_ chr19 49,108,251 49,108,257 TGACCGC > T 1 R376del AKAP13 p.E363del chr15 85,579,148 85,579,151 TGAG > T 1 ATN1 p.E75del chr12 6,934,518 6,934,521 TGAG > T 1 CREB3L4 p.E193del chr1 153,972,773 153,972,776 TGAG > T 1 FMNL2 p.R1071del chr2 152,647,832 152,647,835 TGAG > T 2 GTPBP2 p.L385del chr6 43,624,013 43,624,016 TGAG > T 1 LIPH p.S60del chr3 185,535,002 185,535,005 TGAG > T 1 NAB2 p.E79del chr12 57,091,269 57,091,272 TGAG > T 1 NUDT17 p.E132del chr1 145,846,449 145,846,452 TGAG > T 1 SRCAP p.E2294del chr16 30,736,346 30,736,349 TGAG > T 1 UACA p.L729del chr15 70,668,496 70,668,499 TGAG > T 1 ZFYVE26 p.L2445del chr14 67,752,379 67,752,382 TGAG > T 1 CCAR1 p.R324fs chr10 68,749,527 68,749,531 TGAGA > T 1 KAT6B p.E1355_ chr10 75,028,874 75,028,880 TGAGGAA > T 1 E1356del OSBPL11 p.L302_ chr3 125,563,803 125,563,809 TGATAAA > T 1 S303del URI1 p.D307_ chr19 30,009,233 30,009,239 TGATGAC > T 6 D308del HOXA3 p.R195fs chr7 27,108,661 27,108,663 TGC > T 1 UTRN p.K1448fs chr6 144,491,004 144,491,008 TGCAA > T 1 CCDC102A p.R394del chr16 57,518,132 57,518,135 TGCC > T 1 PDX1 p.P243del chr13 27,924,562 27,924,565 TGCC > T 2 HNRNPDL p.P34_ chr4 82,429,581 82,429,590 TGCCGCGGCG > T 1 R36del NCL p.F690_ chr2 231,455,244 231,455,256 TGCCTCCTCGGAA > T 1 G693del KEHE21 p.A214fs chr1 6,602,171 6,602,176 TGCGCG > T 1 HEXIM1 p.R323_ chr17 45,150,155 45,150,161 TGCGGGA > T 2 E324del SH3D19 p.A93fs chr4 151,175,078 151,175,085 TGCTTCCC > T 1 *HGVS, human genome variation society, http://www.hgvs.org Reference sequence > Altered sequence

More examples include, but are not limited to, the hotspots identified in the following:

Cancer Discov. 2018 February; 8(2): 174-183 (Supplementary Material—Refer to Web version on PubMed Central for supplementary material); Database: The Journal of Biological Databases and Curation, 2020, 1-8; npj Genomic Medicine (2021) 6, Article number: 33; Computational and Structural Biotechnology Journal, Volume 18, 2020, Pages 3567-3576.

As used herein, the nearby region of the hotspot includes DNA with sequence 40 base pair upstream and 40 base pair downstream of the hotspot.

As used herein, Tier 1 5hmC are cytosine (C) residues that exhibit 3 to 8-fold more likelihood of becoming 5hmCs in genomic DNAs from tumor-cells than from normal-cells, and Tier 2 5hmC are sites that exhibit equal allele frequency of 5hmC in both normal and tumor-cells.

As used herein, the genomic DNA includes total or partial full-length or fragmented (i.e., cell-free DNA) genomic DNA isolated from any human tissues, including plasma.

The term “genome” generally refers to an entirety of an organism's hereditary information. A genome can be encoded either in DNA or in RNA. A genome can comprise coding regions that code for proteins as well as non-coding regions. A genome can include the sequence of all chromosomes together in an organism. For example, the human genome has a total of 46 chromosomes. The sequence of all of these together constitutes a human genome.

The term “subject” and “patient” are used interchangeably herein, and refer to an animal, for example, a human from whom cells can be obtained. The term “mammal” is intended to encompass a singular “mammal” and plural “mammals,” and includes, but is not limited to humans; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs; and bears. In some preferred embodiments, a mammal is a human.

The term “sample” as used herein relates to a material or mixture of materials, typically, although not necessarily, in liquid form, containing one or more analytes of interest. Nucleic acid samples may be complex in that they contain multiple different molecules that contain sequences. Genomic DNA from a mammal (e.g., mouse or human) are types of complex samples. Any sample containing nucleic acid, e.g., genomic DNA made from tissue culture cells or a sample of tissue, may be employed herein.

Using chemical oxidation and reduction technique combined with Next Generation Sequencing (NGS), the present inventors explored the existence of 5hmC at cytosine and 5′-C-phosphate-G-3′ (CpG) sites within the gene bodies of a group of oncogenes, especially at or near (e.g., within 40 base pairs) the known cancer mutation hotspots. The cancer mutation hotspot can be expressed as a single base on genomic DNA that is frequently observed to have single nucleotide variant (SNV). The present inventors found that 5hmC does not randomly exist on all CpG sites on a gene, but rather on a small portion of all the CpG sites or cytosine residues. They exist specifically at cytosine sites (mostly at cytosine in CpG islands) located right at or within a range of 40 base pairs of a cancer mutation hotspot. Sometimes 5hmC occurs on a cytosine (C) that is not adjacent to a guanine (G). The results show the presence of two characteristically distinct 5hmC groups: Tier 1 Group with 3 to 8-fold more 5hmCs detected in tumor-cells than in normal-cell derived DNA. Tier 2 group with equal allele frequency of 5hmc among normal and tumor-cell derived DNA at 5 CpG hotspot sites as well as 5 non-CpG hotspots. Significantly more Tier 1 group 5hmC sites are found at hotspots in either tumor cells or cell lines rendered immortal (by transforming agents such as SV40 T-antigen (Simian Vacuolating Virus 40 TAg)) than in healthy normal cells.

FIGS. 1A-1C and 2A-2D illustrates examples of individual 5hmc sites as specific cancer marker (Tier 1) or not as marker (Tier 2) i.e., Tier 1 and Tier 2 Group 5hmCs at or near hotspots.

In particular, FIGS. 1A-1C are a representative Tier 1 observation (arrow 102) at cancer mutation hotspot ERBB4 R711C (Chr2: 211623993).

In FIGS. 1A-1C, the top half of the plots are for untreated, background level of hot spot mutation. The bottom half shows the treated group. Y-Axis: Allele Frequency (AF) shown as fraction of C>T mutation at all observed hotspots. X-Axis: genome coordinates of all nearby CpG sites. Vertical Arrows: Hotspot Location (Vertical arrows 101: Wildtype C or G; Vertical arrows 102: Mutation T or A); Arrows 103 (Tier 2 site): 5hmc detected in DNA from all cells; and Arrows 104 (Tier 1 site near hotspot): 5hmC detected in cancerous cells at non-hotspot CpG sites.

FIGS. 2A-2D illustrate_several more examples of Tier 1 group. In FIGS. 2A-2D, Arrows 202 identify location of Tier 1 5hmc: at or near cancer mutation hotspots; Y-Axis: Allele Frequency (AF) shown as fraction of C>T mutation at all observed hotspots; X-Axis: genome coordinates of nearby CpG sites; Arrows 203 identify location of Tier 1 5mhc not at hotspot; and Arrows 204 identify location of Tier 2 5hmc detected in DNA from all cells.

Allele frequencies (AF %) of detected 5hmC at each cancer mutation hotspot (17 Hotspots) after the treatment are shown in Table 2 and Table 3. Examples of Tier 1 group 5hmC at cancer mutation hotspots (>8% are in bold) are listed in Table 2. Examples of Tier 2 group 5hmC at cancer mutation hotspots (>8% are in bold) are listed in Table 2.

TABLE 2 C of G of C of G of C of G of Tier 1 Bases CpG CpG CpG CpG CpG CpG Chromo- Hotspot Mutation Hotspot at Normal Normal Immprtalized Immprtalized Cancer Cancer some Location name Mutation hotspot (PBMC) (PBMC) (PAM3005) (PAM3005) (HCT116) (HCT116) chr11 108247071 ATMR337C, C > T CpG  2.1% 56.8%  0.7%  8.1% c.1009C > T     chr19 11021837 SMARCA4T910M, C > T CpG 2.0%  6.1%  1.1%  2.2% 10.7% c.2729C > T       chr2 208248388 IDH1R132H, C > T CpG 1.4%  2.0%  9.1%  2.8%  0.5% c.395G > A     chr1 162776212 DDR2R709, C > T CpG 7.1% 0.3%  8.9%  9.6%  7.4% 20.5% c.2125C > T chr17 39711955 ERBB2S310F, C > T CpG 3.8%  12.5%  16.6% c.929C > T chr2 211623993 ERBB4R711C, G > A CpG 1.3% 2.1%  8.5% 25.3%  9.9%  7.8% c.2131C > T chr12 25245350 KRASG12C, C > A CC 22.58% 32.14% c.34G > T

TABLE 3 C of G of C of G of C of G of Tier 2 Bases CpG CpG CpG CpG CpG CpG Chromo- Hotspot Mutation Hotspot at Normal Normal Immprtalized Immprtalized Cancer Cancer some Location name Mutation hotspot (PBMC) (PBMC) (PAM3005) (PAM3005) (HCT116) (HCT116) chr17 7673802 TP53R273H, C > T CpG 1.2% 4.1% 2.5% 4.8% 5.0%  3.3% c.818G > A chr17 7674220 TP53R248Q, C > T CpG 5.8% 0.6% 8.7% 0.4% 5.5%  2.4% c.743G > A chr7 55181378 EGFRT790M, C > T CpG 7.4% 4.8% 6.4% 6.0%  0.7% c.2369C > T chr17 7675088 TP53R175H, C > T CpG 2.3% 4.8% 0.8% 6.3% 3.4%  6.6% c.524G > A chr5 112839941 APCR1450, G > A CpG 6.3% 6.7% 18.6% c.4348C > T chr15 66436824 MAP2K1P124S, C > T CC 7.2% 5.7% 3.8% c.370C > T chr14 104780214 AKT1E17K, C > T CC 2.8% 1.9% 4.7% c.49G > A chr3 41224646 CTNNB1S45F, C > T TCT 2.1% 1.5% 1.0% c.134C > T chr3 179218303 PIK3CAE545K, G > A TGA 2.6% 4.2%  5.4% c.1633G > A chrX 71119404 MED12G44D, G > A GG 5.1% 5.4%  5.9% c.131G > A

DNAs from normal cells (PBMC) and the two cancerous/tumor cells are compared. Both base C and G of the CpG are checked. AFs higher than 8% are shown in bold and those between 4% and 8% can also be noted. In cancerous cells, most CpG hotspot sites have both the C and G in the CpG island mutated. One of the non-CpG hotspot, KRAS G12C (a “CC”, with a C to A mutation), showed significantly more 5hmCs in cancerous cells than in normal cells.

The observations in Table 1 and 2, averaged AF % for each group, before or after the 5hmC>T conversion are plotted in FIG. 3 . Significantly higher AF % are observed in both PAM3005 (transformed cells) and HCT116 (cancer cell lines) in Tier 1, while the AF % were comparable among Tier 2. Background level of AF % for all groups are comparable.

In an expanded studies covering 33 cancer mutation hotspots employing 12 normal and 12 colorectal cancer samples further confirmed the above results in cell culture cells. Significantly more 5hmC sites were observed in tumor than normal DNA at higher AF. For example, at 5% AF or above, an average of 609 5hmC sites were found in each tumor DNA versus 479 in normal DNA. At 10% or higher, the average number was about 153 in tumor versus 66 in normal. The number of extra 5hmC (Tier 1) found in tumor was proportionally higher in high AF range. Calculated as percentage of 5hmC sites found in normal, there were 2%, 36%, 170%, and 283% more 5hmC counts in tumor, and 24%, 46%, 147% and 230% higher sum of AF values in tumor than in normal gDNA, when detection criteria of AF were set at above 1%, 5%, 10 and 12%, respectively (See, FIG. 4 ).

FIG. 4 . Illustrates 5hmC sites in tumor as percentage of 5hmC in normal at increasing AF cut-off.

Tier 1 5hmC sites showing three-fold or higher AF in colorectal tumor cells than in normal colon cells (in the 80 bp hotspot flanking regions studied) are listed in Table 4 (Cancer Hotspot Targets with Single Nucleotide Variant (SNV) below. About half of these sites coincide with known mutation hotspots. Table 4 does not include all Tier 1 sites that are not detected in the experiment nor all Tier 1 sites in cells from other tumor types.

TABLE 4 Chromosome Genomic Location (Hg38) Base Gene chr1 114713883 G NRAS chr1 114713893 G NRAS chr1 114713898 G NRAS chr1 114713917 G NRAS chr1 114713930 C NRAS chr1 114713945 C NRAS chr1 162776178 C DDR2 chr1 204537412 G MDM4 chr1 204537448 C MDM4 chr1 204537462 C MDM4 chr2 211623981 C ERBB4 chr3 179234268 G PIK3CA chr3 41224650 G CTNNB1 chr3 41224652 G CTNNB1 chr4 1801808 G FGFR3 chr4 1801845 G FGFR3 chr4 1801863 G FGFR3 chr4 54285923 G PDGFRA chr4 54727443 G PDGFRA chr5 1295184 G TERT chr5 1295192 G TERT chr6 152098823 C ERSR1 chr7 140753310 G BRAF chr7 55174013 G EGFR chr7 55174016 C EGFR chr7 55174021 G EGFR chr7 55174025 G EGFR chr7 55174048 G EGFR chr7 55174049 G EGFR chr7 55174765 C EGFR chr7 55174769 C EGFR chr7 55181349 C EGFR chr7 55181355 C EGFR chr7 55181361 C EGFR chr7 55181364 C EGFR chr7 55181371 C EGFR chr7 55181382 G EGFR chr7 55191810 C EGFR chr7 55191856 C EGFR chr9 136504900 G NOTCH1 chr9 21971077 C CDKN2A chr10 87933135 G PTEN chr10 87933139 G PTEN chr10 87933144 G PTEN chr10 87933147 C PTEN chr10 87933148 G PTEN chr12 132676624 C POLE chr12 68828834 C MDM2 chr12 68828841 C MDM2 chr13 32332602 C BRCA2 chr14 104780182 G AKT1 chr14 104780194 C AKT1 chr14 104780230 G AKT1 chr14 104780233 G AKT1 chr15 66436832 C MAP2K1 chr15 66436838 C MAP2K1 chr15 66436841 C MAP2K1 chr15 66436853 C MAP2K1 chr17 39531209 G CDK12 chr17 39531210 G CDK12 chr17 39531225 G CDK12 chr17 7673775 C TP53 chr17 7673804 C TP53 chr17 7673836 C TP53 chr17 7674201 G TP53 chr17 7674204 G TP53 chr17 7675067 C TP53 chr17 7675071 G TP53 chr19 11021799 C SMARCA4 chr19 11021822 G SMARCA4 chr19 11021838 G SMARCA4 chr19 1223136 G STK11 chr19 1223144 C STK11 chr19 1223165 G STK11 chrX 77558831 G ATRX

The association of increased quantity of specific, individual 5hmC at or near specific Tier-1 hotspots in cancer cells provides a way to distinguish cancer cells from normal cells directly at specific base (C or G) resolution. Because 5hmC is not detected by normal sequencing technique as mutated, the increased 5hmC occurrence at specific hotspots is a more sensitive marker of cancerous cells before the occurrence of many mutations (e.g., C to T changes). Furthermore, the detection of these specifically selected, individual Tier-1 5hmC sites at or near hotspot CpG sites in cancer cell can be a more convenient, more direct cancer detection method than analysing the group 5hmC profile at chromosomal level or from hundreds of sequences of entire genes.

Thus, the detection and quantification of the number of selected specific individually targeted Tier-1 5hmC sites or its prevalence at or near many cancer mutation hot spots in a given cell enables one to detect, screen and predict the likelihood of cancer occurrence or the severity of the cancer. Moreover, the existence of 5hmC at many hotspots in cancer cell lines suggests a previously unknown higher order mechanism underlying the development of cancer. Markers along the 5hmC-mediated mechanism or pathway in cancer development are not only better diagnostic targets than mutations at hotspots, but also potentially better therapeutic targets. Drugs directly or indirectly either prevent 5hmC from occurring, prevent 5hmC from being converted to uracil- or thymine-analog, or correct 5hmC back to regular cytosine may prevent or treat cancer.

In one aspect, the present disclosure provides a method which includes:

extracting genomic deoxyribonucleic acid (DNA) from locations at or near specific target cancer hotspots from a subject;

modifying specific Tier-1 5-hydroxymethylcytosine (5hmC) on the DNA to a modified specific Tier-1 5hmC;

detecting and identifying presence or absence of the modified specific Tier-1 5hmC;

quantifying the detected and identified modified specific Tier-1 5hmC; and

providing a report comprising a score, wherein the score is indicative of the likelihood of a status, a degree, or a severity of the risk of cancer.

In one embodiment of this aspect, the specific Tier-1 5hmC can exist in cancer cell lines, in transformed and immortalized cells.

In particular, the present disclosure provides selected specific Tier-1 5-hydroxymethylcytosine (5hmC) at or near cancer mutation hot spots as targets for early cancer detection. Such methods provide for high sensitivity detection of one or more genetic variants.

In another embodiment, the method comprises quantifying the detected and identified specific Tier-1 5hmC at or near cancer mutation hot spots located at a specific set of oncogenes in which, when mutated, a cytosine (C) is mutated to thymine (T), or a Guanine (G) is mutated to Adenine (A) on the complementary strand after amplification.

A cancer mutation hot spot is any single nucleotide having substitution mutations reported in the literature that is associated with any cancer. The cancer mutation hotspot can also be expressed as a single base on genomic DNA that is frequently observed to have single nucleotide variant (SNV) or deletion.

In another embodiment, modifying specific Tier-1 5hmC on the DNA to a modified 5hmC includes treating genomic deoxyribonucleic acid (DNA) to convert 5hmC on the DNA to a modified 5hmC includes any technique to modify 5hmC into another derivative of a nitrogenous base, such as derivative of a cytosine (C) or a thymine (T), or any non-nitrogenous molecule which can be detected as a different base from the original 5hmC. The detected different base can be used to calculate the quantity of 5hmC at any specific nucleotide locations on human genome.

In another embodiment, treating genomic deoxyribonucleic acid (DNA) to convert specific Tier-1 5hmC on the DNA to a modified 5hmC includes a method that employs either chemical or enzymatic reaction processes or both to modify the 5hmC into another derivative of a nitrogenous base, such as derivative of a cytosine (C) or a thymine (T), or any non-nitrogenous molecule which can be detected as a different base from the original 5hmC.

In another embodiment, treating genomic deoxyribonucleic acid (DNA) to convert specific Tier-1 5hmC on the DNA to a modified 5hmC includes a method that employs either oxidation or reduction reaction processes or both to modify the 5hmC into another derivative of a nitrogenous base, such as derivative of a cytosine (C) or a thymine (T), or any non-nitrogenous molecule which can be detected as a different base from the original 5hmC (C). In preferred embodiments, the oxidation or reduction reaction processes can be either chemical or enzymatic reactions.

Preferably, the oxidising agent may be an organic or inorganic chemical compound. Suitable oxidising agents are well known in the art and include metal oxides, such as Potassium perruthenate (KRuO4), Manganese dioxide (MnO2), Potassium permanganate (KMnO4). Particularly useful oxidising agents are those that may be used in aqueous conditions. However, oxidising agents that are suitable for use in organic solvents may also be employed where practicable. In some embodiments, the oxidising agent may comprise a perruthenate anion (RuO). Suitable perruthenate oxidising agents include organic and inorganic perruthenate salts, such as potassium perruthenate (KRuO4) and other metal perruthenates; tetraalkylammonium perruthenates, such as tetrapropylammonium perruthenate (TPAP) and tetrabutylammonium perruthenate (TB AP); polymer-supported perruthenate (PSP) and tetraphenylphosphonium ruthenate.

Advantageously, the oxidising agent or the oxidising conditions may also preserve the DNA in a denatured state. Optionally, the polynucleotide (DNA) may be subjected to further, repeat oxidising steps.

Suitable reducing agents are well-known in the art and include Pic-borane, Pyridine borane, Sodium borohydride (NaBH4), Sodium cyanoborohydride (NaCNBH4) and Lithium borohydride (LiBH4). Particularly useful reducing agents are those that may be used in aqueous conditions, as such are most convenient for the handling of the polynucleotide (DNA). However, reducing agents that are suitable for use in organic solvents may also be employed where practicable.

In another embodiment, the method further includes any technique for one of more of capturing, sequestering and enriching DNA fragments of 1000 base pair or less from any human tissue or cells by any molecule, such as monoclonal or polyclonal antibodies, having specific affinity in binding to specific Tier-1 5hmC. The captured, sequestered, or enriched DNA can be then analyzed to calculate the quantity of a variable which is a function of the quantity of cancer-specific genetic features, which include but not limit the quantity of cancer mutation hotspots.

In another embodiment, the method employs a method to quantify the number of detected specific Tier-1 5hmC occurred at or near a specific hotspot or multiple of hot spots or one or more cytosine near the hotspot.

In another embodiment, the present disclosure comprises any anti-cancer therapeutic methods or agents targeting either the specific Tier-1 5hmC itself, biochemical steps of converting regular cytosine to 5hmC, conversion of the 5hmC to uracil- or thymine-analog, or the 5hmC-mediated pathway that leads to cancer development.

In another embodiment, the method comprises any reference material, including but not limited to primary standard, secondary standard, calibrator, quality control, validation sample, using any of the specific Tier-1 5hmC at hotspot and its nearby region as part of the reference DNA sequence composition for diagnosis of cancer via specific Tier-1 5hmC detection, and quantification.

In another embodiment, the method includes quantifying a variable which is a function of a quantity of specific Tier-1 5-hydroxymethylcytosine (5hmC) at any specific nucleotide location on a human genome; and thereby detecting, screening or predicting a likelihood of cancer occurrence in a subject.

In another embodiment, the method provides the diagnostic methods that comprises the following steps:

Step 1: Modification of specific Tier-1 5hmC at locations which are at or near the said cancer hotspots.

Genomic DNA from human tissue (including plasma) is pre-extracted from patient specimen. It is subjected to a treatment to convert 5hmC on the DNA to a different moiety, such as an uracil, that is recognizable to identify the location of the 5hmC.

Examples of modification methods comprise the following:

-   -   (1) DNA containing 5hmC or the cancer hotspots and its adjacent         region is oxidized by potassium perruthenate (KRuO₄) or other         salts of high oxidation state of transition metals such as         potassium permanganate (KMnO₄), or other oxidizing agent, to         produce an aldehyde, such as 5-formylcytosine (5fC). 5fC is then         reduced by a reducing agent such as Pic-borane or Pyridine         borane to produce an uracil derivative dihydrouracil (DHU). DHU         is then recognized as thymine (T) in the subsequence replication         or amplification reaction involving RNA and/or DNA polymerase         and any DNA sequence identification method. Alternatively,         cytosine (C)'s complementary base, guanine (G) is recognized as         Adenine (A) after replication or amplification reaction         involving RNA and/or DNA polymerase and any DNA sequence         identification method.     -   (2) DNA containing 5hmC or the cancer hotspots and its adjacent         region is oxidized by enzymes such as Ten-eleven translocation         (TET)1, TET2, and TET3, or another oxidative enzyme modifying         5hmC. The product of the oxidation of 5hmC is 5fC and         subsequently 5-carboxylcytosine (5caC). These products are then         reduced by reduction agents such as bisulfite (NaHSO₃) and         produce derivative of uracil which is subsequently recognized as         thymine (T) in the subsequence replication or amplification         reaction involving RNA and/or DNA polymerase. Alternatively,         cytosine (C)'s complementary base, guanine (G) is recognized as         Adenine (A) after replication or amplification reaction         involving RNA and/or DNA polymerase and any DNA sequence         identification method. In addition to replication or         amplification, C-to-T change or G-to-A change at the hotspot can         be recognized (and distinguished from other nucleotides) by         other methods disclosed in (1).     -   (3) The chemicals or enzymes in oxidations and reductions in (1)         and (2) can be optionally switched to achieve the same modifying         result, i.e., either C is converted to T, or after replication,         its complementary base G is converted to A.     -   (4) Different modifications using oxidation or reduction can be         applied to regular cytosine base (C), 5-methylcytosine (5mC),         and 5hmC separately in order to produce different products so         that the three can be distinguished and identified in subsequent         procedures. For example, bisulfite reaction can distinguish         regular cytosine from 5mC and 5hmC by modifying the regular         cytosine. Alternatively, TET can separate both 5mC and 5hmC from         regular cytosine by modifying both 5mC and 5hmC. In a separate         experiment, 5mC and 5hmC can be distinguished by protection of         5hmC specifically from oxidation and reduction by using         β-glucose transferase to attach glucose to the hydroxyl group of         5hmC to create 5-glucosyl-hydroxylmethylcytosine (5-ghmC). The         unprotected 5mC can be reduced to produce DHU while the same         reaction is blocked on 5-ghmC.     -   (5) Alternatively, regular cytosine, 5mC and 5-ghmC can be         distinguished by their susceptibility to restriction digestion         by enzymes such as MspI and HpaI.     -   (6) Specific sequence guided or sequence dependent recognition         or cutting of DNA in the vicinity of regular cytosine, 5mC and         5-ghmC at or near a cancer mutation hotspot is performed via         techniques such as DNA- or RNA-guided gene editing (such as         Crisper technology), homologous recombination, or transposition         via transposon.

Step 2: Detection, identification or confirmation of the presence or absence of modified 5hmC at specific Tier-1 locations which are at or near the said cancer hotspots.

Example

-   -   (1) The DNA region having the modified specific Tier-1 5hmC is         replicated, amplified or copied by DNA or RNA polymerase, the         modified bases (from Step 1) contribute to the identification of         the 5hmC and its location by being recognized by the polymerase         as a different deoxy-ribonucleotide such as thymine (T, for         modified C) or adenine (A) on its complementary strand.     -   (2) The DNA region having the protected specific Tier-1 5hmC         (such as 5-ghmC) is replicated, amplified or copied by DNA or         RNA polymerase as regular cytosine, while regular cytosine and         other cytosine derivative such as 5mC are recognized as a         different deoxy-ribonucleotide such as thymine (T) or         adenine (A) on its complementary strand.     -   (3) The detection methods of (1) and (2) comprise various         processes of replication or amplification mediated by DNA or RNA         polymerase. These methods comprise, Sanger Sequencing, massive         paralleled sequencing or Next Generation Sequencing (NGS), any         form of single-cell-sequencing, such as technologies from         Polymerase Chain Reaction (PCR), Droplet Digital PCR (ddPCR),         Quantitative PCPacific Biosciences, Oxford Nanopore Technology,         Quantapore (CA-USA), and Stratos (WA-USA), R (qPCR), Reverse         Transcription PCR, isothermal amplification.

As examples shown in FIGS. 1A-1C, 2A-2D, 3 and 4_and Table 2 and 3, number of reads of 5hmC was obtained by NGS, and allele frequency (AF) can be calculated reflecting the frequency or amount of the detected 5hmC. The detection signal of Tier-1 5hmC can be generated by a single 5hmC site or multiple specific Tier-1 5hmC sites.

-   -   (4) The detection methods of (1) and (2) can include RNA-guided         gene editing methods.     -   (5) Regular C, 5mC, 5hmC and their modified forms generated in         Step 1 can be distinguished from each other by using different         restriction enzymes that exhibit differential cutting efficiency         among modified or unmodified forms. With or without PCR         amplification, the size pattern of the restriction product can         be compared using agarose gel or any form of chromatography.     -   (6) The detection methods of (1) and (2) can employ any         technique to capture, sequester or enrich DNA fragments of 1000         base pair or less from any human tissue or cells by any         molecule, such as monoclonal or polyclonal antibodies from any         species, having specific and affinity in binding to 5hmC.     -   (7) In addition to replication or amplification, C-to-T change         or G-to-A change at the hotspot can be recognized and         distinguished from other nucleotides by other methods comprise         chromatographical methods (e.g., size exclusion, affinity         binding, ion-exchange chromatography), mass spectrometry,         affinity binding and labelling methods utilizing         antigen-antibody interaction (such as in ELISA), and         molecule-to-molecule affinity binding (such as ligand-receptor         binding).

Another example of detection method:

-   -   1) DNA oligos (primers) containing DNA sequence at or adjacent         to Tier 1 sites were synthesized. These probes can be either         immobilized on solid surface (flat or non-flat such as a         magnetic bead surface) or chemically cross-linked to a moiety         that is able to bind to a solid surface via affinity binding.     -   2) Labeled DNA oligos (probes) containing DNA sequence including         one or multiple Tier 1 sites and sequences adjacent to them are         synthesized. The deoxyribonucleotide at the Tier 1 5hmC location         is a T (or A for the base complementary to 5hmC). This allows         the probe to specifically hybridize to modified 5hmC after it is         modified to uracil and subsequently amplified as T. The probe         can serve as reporter (marker) during subsequent detection step         (6).     -   3) The liquid biopsy sample (either from plasma or other body         fluid) consists of numerous cell free DNA (cfDNA) derived from         genomic DNA from either normal or tumor cells. The total cfDNA         can be extracted from the sample using a variety of methods.     -   4) After extraction, cfDNA is subject to 5hmC modification         (described in Step 1).     -   5) The pre-synthesized primers (oligos) from 1) are subjected to         contacting with the modified extracted cfDNA (from 3)) via         mixing or incubation under specific conditions promoting         denaturation of the double stranded DNA, followed by         hybridization of single-stranded DNA molecules based on         complementary pairing scheme (ie. A to T, C to G).     -   6) The hybridized DNA is pulled out from the mixture via         affinity binding followed by washing. This step can be skipped         if enough DNA containing Tier 1 sites is available for analysis.         If there is insufficient Tier 1 collected, multiple rounds of         steps 2) to 5) can be done to accumulate sufficient DNA         containing Tier 1 sites.     -   7) The probe from 2) is mixed with the hybridized DNA from 5) in         a qPCR or ddPCR reaction. The labeled moiety of the probe         provides signal indicating the quantity of the Tier 1 5hmC in         the sample.     -   8) FIG. 5 shows an example amplification plot from qPCR. Nine         Tier-1 5hmC targets were selected from Table 4 for preparing the         assay with primer and probes synthesized. Equal amount of gDNA         from a pool of colorectal cancer patient and their matched         normal colorectal tissue were extracted. Real-time florescence         curves, indicating the real-time detection of 5hmC, were         plotted. Cycle Threshold (Ct) values (26.1 for cancer sample and         31.6 for normal sample), which reflecting the relative         concentration of the 5hmC, can be obtained.

Step 3: Quantification of the detected and identified 5hmC at locations which are at or near the said cancer hotspots.

Quantifying or recording the quantity of the occurrence of 5hmC can be of the following forms:

-   -   (1) Absolute number, count, read, or event of such 5hmC found in         a given sample preparation.     -   (2) Absolute number, count, read, or event of such 5hmC detected         on one or more specific genes in any given sample preparation.         The quantified number can be either from a single specific         Tier-1 5hmC or multiple Tier-1 5hmCs.     -   (3) Relative allele frequency or ratio or percentage of absolute         numbers of 5hmC relative to either regular cytosine (C) or         combination of regular C, 5mC and 5hmC at the same allele (base         location) in situations in (1) and (2).     -    As examples shown in FIGS. 1A-1C and 2A-2D, 3 and 4 , and Table         1 and 2, allele frequency (AF) was calculated indicating the         quantification of the 5hmC based on the number of reads of 5hmC         obtained by NGS.     -   (4) Relative numbers derived, transformed, or calculated from         signal (e.g., florescence index), absorbance, intensity, color,         hue, area of peak, or other measurements which reflect the         numbers in (1), (2), or (3).     -    As an example in FIG. 5 , difference in Ct values (DeltaCt) can         be calculated to indicate the degree of 5hmC concentration         difference. In addition, average, sum, square, exponential         power, differences, ratio, or other simple mathematical         operation or transformation that are used to reflect the         quantity of the detected and identified specific Tier-1 5hmC at         locations which are at or near the said cancer hotspots.

Step 4: The quantity of the quantitated number in Step 3 is applied to a predetermined algorithm so that a score is generated that is comparable to predetermined criteria that is indicative of the status, degree, severity, or size of the risk of cancer of that patient.

Examples

-   -   (1) A score is a calculated value of a variable that is         measuring of the propensity or likelihood of a patient's chance         in getting cancer (or severity of cancer). In examples shown in         FIG. 4 , the score is a percentage calculated between AF of         detected 5hmC in cfDNA versus gDNA from normal tissue.     -   (2) In example of FIG. 5 , the score can either be the deltaCt         or the deltaCt value can be converted into a ratio between         concentrations of targeted Tier-1 5hmC in tumor and normal         tissue. In this case, the ratio is 44.8.

The score calculated in (1) and (2) can be compared to a predetermined cut-off value (criteria or limit values, see Step 5) to determine the presence of tumor.

Step 5: Via mass observations (clinical trials) on a population of normal and pre-cancer or cancer patient samples, steps 1, 2 and 3 are used to generate raw data for generating an algorithm.

-   -   (1) The algorithm is a mathematical relationship between the         quantified specific Tier-1 5hmC values (obtained in Step 3) and         a score representing the degree of likelihood of having cancer.     -   (2) The score representing the likelihood of cancer can be         obtained by giving a severity number to each patient based on         the patient's size of tumor or stages of cancer.     -   (3) Regression models may be established between the quantified         specific Tier-1 5hmC values (obtained in Step 3) and the score         representing the likelihood of cancer.     -   (4) Based on large population of data, the cut-off value, the         score that can separate normal or cancer patient can be         statistically determined.

In another embodiment, the present disclosure provides both the Tier1 and Tier2 5hmC sites as targets for making contrived patient-like reference materials, including positive or negative quality control samples, standards (eg. a primary standard, a secondary standard, or a calibrator), or validation samples for assays aiming for detecting Tier1 or Tier2 5hmC to detect cancer. Synthetic DNA fragments mimicking the 5hmC patterns (at Tier1 or Tier2 sites) in genomic DNAs from either tumor cells or normal cells can be produced either through DNA synthesis in vitro or site-directed gene-editing in vivo. The resulting contrived sample can be used to monitoring the performance of the assay or calibrating the measurement system within the assay.

In another embodiment, the present disclosure provides anti-cancer therapeutic methods targeting Tier-1 5hmC at or near hotspot that comprises the following strategies:

-   -   (1) Methods or agents preventing the conversion from regular         cytosine to 5hmC at or near cancer mutation hotspots.

Many biochemistry processes or pathways exist that result in 5hmCs, specifically located at or near cancer mutation hotspot, from regular cytosine or an intermediate, such as 5mC.

For example, enzymes Ten-eleven translocation (TET)1, TET2, and TET3 catalyzes the conversion of 5mC to 5hmC. Inhibitors of TET can be used to prevent this process. Specifically, any inhibitors that directly or indirectly inhibits the 5hmC formation at or near cancer mutation hotspot to achieve anti-cancer effect are encompassed within the scope of this disclosure.

Alternatively, methods or agents that prevent the formation of 5hmC at or near cancer hotspots through TET-independent mechanisms are also encompassed within the scope of this disclosure.

-   -   (2) Methods or agents preventing the formation of uracil- or         thymine-analog from 5hmC at or near cancer mutation hotspots.

Any methods or agents that directly or indirectly inhibit the cellular process converting 5hmC to uracil- or thymine-analog at or near cancer mutation hotspots are encompassed within the scope of this disclosure.

-   -   (3) Methods or agents converting, directly or indirectly 5hmC to         cytosine or another cytosine derivative (recognized as “C” by         RNA or DNA polymerases) at or near cancer mutation hotspot are         also encompassed within the scope of this disclosure.

All combinations of modification strategies, aimed to identify 5-hmC at locations which are at or near the said cancer hotspots are encompassed within the scope of this disclosure.

The below references cited herein are hereby incorporated by reference-for-all purposes.

REFERENCES

-   Pfeifer G P, et al. 5-hydroxymethylcytosine and its potential roles     in development and cancer. Epigenetics & Chromatin. 2013; 6: 10. -   Singh A K, et al. Selective targeting of TET catalytic domain     promotes somatic cell reprogramming. PNAS. 2020; 117: 3621-3626. -   Gerecke C, et al. Vitamin C in combination with inhibition of mutant     IDH1 synergistically activates TET enzymes and epigenetically     modulates gene silencing in colon cancer cells. Epigenetics 2020     March; 15(3): 307-322 -   Bai X, et al. Ten-Eleven Translocation 1 promotes malignant     progression of Cholangiocarcinoma with wild-type isocitrate     dehydrogenase 1. Hepatology. 2021 May; 73(5): 1747-1763 -   Margalit S, et al. 5-Hydroxymethylcytosine as a clinical biomarker:     Fluorescence-based assay for high-throughput epigenetic     quantification in human tissues. Int. J. Cancer. 2019; 146: 115-122. -   Li W, et al. 5-Hydroxymethylcytosine signatures in circulating     cell-free DNA as diagnostic biomarkers for human cancers. Cell     Research. 2017; 27: 1243-1257. -   Zeng C, et al. Towards precision medicine: advances in     5-hydroxymethylcytosine cancer biomarker discovery in liquid biopsy.     Cancer Communications. 2019; 39: 12. -   Song C X, et al. Mapping recently identified nucleotide variants in     the genome and transcriptome. Nat. Biotechnol. 2012; 30: 1107-1116. -   Yu M, et al. Base-resolution analysis of 5-hydroxymethylcytosine in     the mammalian genome. Cell. 2012 Jun. 8; 149(6): 1368-80 -   Nestor C E, et al. Tissue type is a major modifier of the     5-hydroxymethylcytosine content of human genes. Genome Res. 2012;     22: 467-477. -   Thomson J P, et al. The application of genome-wide     5-hydroxymethylcytosine studies in cancer research. Epigenomics     2017; 9: 77-91. -   Han D, et al. A highly sensitive and robust method for genome-wide     5hmC profiling of rare cell populations. Mol Cell. 2016; 63:     711-719. -   Chen K, et al. Loss of 5-hydroxymethylcytosine is linked to gene     body hypermethylation in kidney cancer. Cell Res. 2016; 26: 103-118. -   Vasanthakumar A, et al. 5-hydroxymethylcytosine in cancer:     significance in diagnosis and therapy. Cancer Genet. 2015; 208:     167-177. -   Li X, et al. Whole-genome analysis of the methylome and     hydroxymethylome in normal and malignant lung and liver. Genome Res.     2016; 26: 1730-1741. -   Köhler F, et al. DNA Methylation in Epidermal Differentiation,     Aging, and Cancer. J Invest Dermatol. 2020; 140: 38-47. -   Sholl L M, et al. The Promises and Challenges of Tumor Mutation     Burden as an Immunotherapy Biomarker: A Perspective from the     International Association for the Study of Lung Cancer Pathology     Committee. J Thorac Oncol. 2020; 15: 1409-1424. -   Constâncio V, et al. DNA Methylation-Based Testing in Liquid     Biopsies as Detection and Prognostic Biomarkers for the Four Major     Cancer Types. Cells 2020; 9(3):624. -   Addeo A, et al. TMB or not TMB as a biomarker: That is the question.     Crit Rev Oncol Hematol. 2021; 163: 103374 -   Arensdorf P, et al. Pancreatic Ductal Adenocarcinoma Evaluation     Using Cell-free DNA Hydroxymethylation Profile. U.S. Patent     Publication No. 2021/0108274.

The above disclosure of this invention is directed primarily to embodiments and practices thereof. It will be readily apparent to those skilled in the art that further changes and modifications in actual implementation of the concepts described herein can easily be made or may be learned by practice of the invention, without departing from the spirit and scope of the invention as defined by the following claims. 

We claim:
 1. A method comprising: extracting genomic deoxyribonucleic acid (DNA) from locations at or near cancer hotspots from a human; modifying specific Tier-1 5-hydroxymethylcytosine (5hmC) on the DNA to a modified specific Tier-1 5hmC; detecting and identifying presence or absence of the modified specific Tier-1 5hmC; quantifying the detected and identified modified specific Tier-1 5hmC; and providing a report comprising a score, wherein the score is indicative of the likelihood of a status, a degree, or a severity of the risk of cancer, wherein the specific Tier-1 exist in cancer cell lines, in transformed and immortalized cells.
 2. The method according to claim 1, wherein extracting genomic deoxyribonucleic acid (DNA) comprises pre-extracting the genomic deoxyribonucleic acid (DNA) from a tissue or plasma of the human.
 3. The method according to claim 1, wherein modifying 5hmC comprises modifying 5hmC into a derivative of a nitrogenous base or a non-nitrogenous base which is a different base from unmodified 5hmC.
 4. The method according to claim 1, wherein the modified 5hmC is a derivative of a cytosine (C) or a thymine (T), which is a base different from the unmodified 5hmC.
 5. The method according to claim 1, wherein the specific Tier-1 sites exist in cancerous cells in organs of the human.
 6. The method according to claim 1, wherein the specific Tier-1 sites exist in colon of the human.
 7. The method according to claim 1, wherein the modifying 5hmC comprises modifying 5hmC with chemical reactions, and wherein the chemical reactions comprise oxidation or reduction reaction.
 8. The method according to claim 1, wherein the modifying 5hmC comprises modifying 5hmC with enzymatic reactions.
 9. The method according to claim 1, wherein the modifying 5hmC comprises modifying 5hmC with an oxidation agent or a reduction agent.
 10. The method according to claim 9, wherein the oxidation agent is potassium perruthenate (KRuO₄), manganese dioxide (MnO₂), potassium permanganate (KMnO₄), tetrapropylammonium perruthenate (TPAP), tetrabutylammonium perruthenate (TBAP), polymer-supported perruthenate (PSP), tetraphenylphosphonium ruthenate, or a combination thereof.
 11. The method according to claim 9, wherein the reduction agent is pic-borane, pyridine borane, sodium borohydride (NaBH₄), sodium cyanoborohydride (NaCNBH₄), lithium borohydride (LiBH₄), or a combination thereof.
 12. The method according to claim 1, wherein detecting and identifying comprises replicating, amplifying, or copying the DNA region having the modified specific Tier-1 5hmC by DNA or RNA polymerase.
 13. The method according to claim 12, wherein detecting and identifying further comprises identifying the 5hmC and its location recognized by the DNA or RNA polymerase as a different deoxy ribonucleotide on its complementary strand.
 14. The method according to claim 13, wherein the different deoxy ribonucleotide is cytosine (C) mutated to thymine (T), or a Guanine (G) mutated to Adenine (A) on the complementary strand.
 15. The method according to claim 1, wherein identifying, and detecting comprises capturing, sequestering and enriching DNA fragments having a base pair less than 1000 from a tissue or a cell of the human, monoclonal antibodies, or polyclonal antibodies, wherein the DNA fragments have a specific affinity in binding to 5hmC.
 16. The method according to claim 1, wherein quantifying the detected and identified modified specific Tier-1 5hmC comprises one or more of quantifying or recording the quantity of the occurrence of 5hmC.
 17. The method according to claim 1, wherein the at or near region of the hotspot includes DNA with sequence 40 base pair upstream and 40 base pair downstream of the hotspot.
 18. The method according to claim 1, wherein the method further comprises using a reference material, wherein the reference material is a primary standard, a secondary standard, a calibrator, a quality control, a validation sample.
 19. The method according to claim 17, wherein the method comprises using the reference material for the specific Tier-1 5hmC at cancer hotspots and nearby region as a part of the reference DNA sequence composition for detection, and quantification. 